Emergence of high-level aztreonam–avibactam and cefiderocol resistance following treatment of an NDM-producing Escherichia coli bloodstream isolate exhibiting reduced susceptibility to both agents at baseline

Abstract Background Cefiderocol (FDC) or ceftazidime-avibactam with aztreonam (CZA-ATM) are frontline agents for New Delhi metallo-β-lactamase (NDM)-producing Enterobacterales; however, clinical data are scarce, and mechanisms of treatment-emergent resistance are ill-defined. Our objectives were to characterize serial isolates and stool microbiota from a liver transplant recipient with NDM-producing Escherichia coli bacteraemia. Methods Isolates collected pre- and post-CZA–ATM treatment underwent broth microdilution susceptibility testing and whole-genome sequencing. Longitudinal stool collected during CZA–ATM therapy underwent metagenomic sequencing (Nanopore MinION). Results The baseline isolate exhibited elevated MICs for ATM–AVI (16/4 µg/mL) and FDC (8 µg/mL). Posttreatment, a rectal surveillance isolate exhibited high-level resistance to ATM–AVI (> 128/4 µg/mL) and FDC (32 µg/mL). Both isolates belonged to ST361 and harboured WT blaNDM-5. The baseline isolate contained wild type (WT) blaCMY-145 and mutations in ftsI (which encodes PBP3), including a YRIN insertion at residue 338 and the non-synonymous substitutions Q227H, E353K and I536L. The posttreatment isolate harboured new mutations in ftsI (A417 V) and blaCMY-145 (L139R and N366Y). Analysis of four stool samples collected during CZA–ATM treatment revealed high E. coli abundance. E. coli relative abundance increased from 34.5% (first sample) to 61.9% (last sample). Conclusions Baseline mutations in ftsI were associated with reduced susceptibility to ATM–AVI and FDC in an ST361 NDM-5-producing E. coli bloodstream isolate. High-level resistance was selected after CZA–ATM treatment, resulting in new ftsl and blaCMY-145 mutations. These findings underscore the need for ATM–AVI susceptibility testing for NDM producers, and the potential for PBP3 mutations to confer cross-resistance to ATM–AVI and FDC, which can emerge after CZA–ATM treatment.


Introduction
The novel β-lactam/β-lactamase inhibitors ceftazidime-avibactam (CZA), meropenem-vaborbactam and impenem-relebactam have revolutionized the management of carbapenem-resistant Enterobacterales (CRE) that produce Klebsiella pneumoniae carbapenemases (KPCs). 1 Unfortunately, these agents lack activity against Enterobacterales carrying metallo-β-lactamases (MBLs), including New Delhi metallo-β-lactamases (NDMs), limiting therapeutic options for these organisms.In recent years, two frontline options have emerged for MBL-producing CRE.The first is combination therapy with CZA and aztreonam (CZA-ATM), which is active against MBL-producing CRE because ATM is not hydrolyzed by MBLs, and avibactam (AVI) protects aztreonam from hydrolysis by serine β-lactamases often present in MBL-producing CRE. 1 ATM-AVI is not yet available (NCT03329092), necessitating the use of combination CZA-ATM. 1 Cefiderocol (FDC), a novel siderophore antibiotic, demonstrates excellent in vitro activity against these pathogens. 1][4][5][6][7][8][9] Moreover, treatment-emergent resistance to either agent has not been widely reported. 10,11erein, we report a liver transplant recipient who developed bacteraemia on post-operative day (POD)-6 with an Escherichia coli isolate that produced NDM-5 and CMY-145, with reduced susceptibility to ATM-AVI and FDC.Bacteraemia resolved with CZA-ATM treatment, but the patient later developed rectal colonization with E. coli exhibiting high-level resistance to both agents.We sought to determine the mechanisms of baseline and treatment-emergent resistance and to characterize the stool microbiota during and after CZA-ATM treatment.

Case presentation
A 48-year-old male with alcoholic cirrhosis underwent a living-donor liver transplant from his daughter under basiliximab induction.He received tacrolimus, mycophenolate and prednisone; standard post-operative prophylaxis consisted of trimethoprim-sulfamethoxazole, acyclovir, fluconazole and 5 days of ampicillin-sulbactam.The patient was originally from India and had immigrated to the USA 25 years prior.He visited India periodically; his last trip was 1 year pre-transplant.His daughter had last travelled to India 4 years pre-donation.
The patient's intra-operative course was uncomplicated.On post-operative day (POD)-5, he developed a fever (38.0°C), but was otherwise stable.Blood cultures were collected on POD-6 (Figure 1), and he was started on vancomycin and piperacillin-tazobactam. Cultures grew E. coli in seven of eight bottles.Susceptibility testing (POD-8) revealed resistance to all routinely tested agents, except aminoglycosides.Carbapenemase production was identified using the modified carbapenem activation method. 12eflex susceptibility testing for CZA and meropenem-vaborbactam was requested, and the patient was switched to meropenemvaborbactam (4 g IV every 8 h) for presumed KPC-E.coli.Blood cultures on POD-8 remained positive.Susceptibility testing results revealed that the initial E. coli was resistant to both CZA and meropenem-vaborbactam.An in-house multiplex PCR assay detected bla NDM on POD-9; these results were shared with the clinical team, prompting a switch to CZA 2.5 g IV every 8 h and aztreonam 2 g IV every 8 h.
Six sets of follow-up blood cultures were negative.A bile leak was managed surgically.Computed tomography (CT) did not show intra-abdominal collections.The patient received CZA-ATM for 20 days, through POD-29.On POD-31, he became febrile to 38.2°C but remained stable.Blood and urine cultures were negative for E. coli, although the urine culture grew vancomycinresistant Enterococcus (Figure 1).A CT scan showed a biloma and fluid collections, which were not amenable to procedures.He received an empiric 2-week course of CZA-ATM, through POD-46; collections decreased in size.On POD-91, imaging revealed no biliary leak and resolution of the collections.The patient improved and was discharged, with no relapse of this infection nearly 3 years post-transplant.
Cultures from CRE rectal surveillance swab, which are performed weekly for routine surveillance on all transplant recipients (chromogenic agar, Hardy Diagnostics, Santa Maria, CA, USA), were negative on POD-20, positive for E. coli on POD-29 and negative on POD-48 (Figure 1).A baseline pre-transplant surveillance swab was not available.

Methods
To characterize E. coli isolates and stool microbiota, the patient was enrolled in a prospective observational study at UPMC (STUDY20090146).

Haidar et al. E. coli causing bacteraemia (isolate-A) and rectal colonization (isolate-B)
were collected (Figure 1).Stool samples were not available prior to bacteraemia from the patient or the patient's daughter.Susceptibility testing was performed by broth microdilution in triplicate according to the CLSI guidelines. 13ATM-AVI was tested as a surrogate for CZA plus ATM, and the ATM breakpoint was used to define susceptibility. 14We performed whole genome sequencing (WGS) as previously described. 15Stool from POD-14, 21, 28 and 30 underwent metagenomic sequencing using the Oxford Nanopore Technologies MinION Mk1c device as previously described 16 (Supplementary Methods, available as Supplementary data at JAC-AMR Online).
Isolate-B developed a > 8-fold increased ATM-AVI MIC compared with isolate-A.We hypothesize that this was mediated by new mutations in PBP3 and CMY-145 that were selected during CZA-ATM therapy.Isolate-B also carried a new A417V substitution in PBP3, which lies opposite the PBP3 active site and hinders substrate binding, since valine is bulkier than alanine. 3,8ew L139R and N366Y substitutions in CMY-145 may have further contributed to ATM-AVI resistance due to increased ATM hydrolysis or weaker inhibition by AVI. 4,6DC is another key option for MBL-producing CRE, resulting in numerically lower all-cause mortality versus other therapies in clinical trials of patients whose isolates exhibited FDC MICs ≤4 µg/mL.19 By contrast, our patient's baseline isolate demonstrated a FDC MIC of 8 µg/mL.As with ATM-AVI, the mechanisms of FDC resistance are multifactorial and likely related to mutations in PBP3 and/or CMY.3,17,20 For example, the 333-YRIN insertion in PBP3 and the substitutions Q227H, E349K, I532L and A412V [identified in isolate-A (338 insertion) and/or isolate-B, Table 1] have been associated with FDC resistance.3,20 Co-existence of CMY-145 20 (identified in both our isolates) and mutations within and increased expression of CMY-type AmpC β-lactamases 17 have also been documented in MBL-producing isolates with FDC resistance.The extent to which the new L139R and N366Y mutations in CMY-145 contributed to increased FDC resistance in isolate-B is unknown.Increased expression of bla NDM-5 has been previously associated with FDC resistance in a single case, but unlike our case, PBP3 mutations and bla CMY were not detected. 11Mutations in other genes, including envZ, marR and aroP, have also been reported following FDC treatment of NDM-producing E. coli, but did not vary from baseline in our case.Finally, although mutations in the iron transporter gene cirA have been associated with FDC resistance, 3,20 these were not identified in either case.
Since our patient's bacteraemia resolved with CZA-ATM treatment, and because rectal CRE colonization predicts infection post-transplant, 21 we hypothesized that interrogating the stool microbiome would reveal a reduction in the abundance of E. coli.Instead, analysis of serial stool samples showed a high abundance of E. coli and persistent detection of bla NDM-5 from POD-18 NDM-producing E. coli bloodstream isolate Our report has some limitations, including the fact that our findings are based on a single patient.Nonetheless, our experience corroborates other reports.In addition to the previously mentioned case, 11 Simner et al. 10 also described another patient who underwent kidney transplant in India and then developed pyelonephritis due to an ST167, NDM-5-producing E. coli with reduced susceptibility to ATM-AVI and FDC.Resistance was attributed to the presence of CMY-59 and a YRIN insertion at PBP3 position 338.In our case, the specific contribution of new PBP3 and CMY mutations resulting in higher-level resistance requires future validation.Although we characterized the stool microbiome over a 2-week period, the absence of stool pre-transplant and at additional timepoints post-transplant prevents us from making other conclusions.In addition, sparse sampling from the patient by peri-rectal surveillance swabs limits our ability to comment on the duration of colonization or possibility of decolonization following treatment.Finally, we could not obtain biospecimens from the patient's daughter to determine whether the E. coli was donor-derived.
Our experience and recent reports indicate that the activity of ATM-AVI and FDC should not be presumed against NDM-producing E. coli and that therapy should be guided by susceptibility testing whenever possible.The greatest vulnerability to both these frontline options appears to be the presence of specific PBP3 mutations and CMY β-lactamases; however, other mechanisms of resistance have been reported. 11Future studies should also evaluate whether optimized CZA-ATM dosing may overcome reduced baseline susceptibility or suppress the emergence of resistance, 22 the clinical effectiveness of ATM-AVI against diverse MBL-CRE, and the role of novel agents 10 such as FEP-zidebactam, FEP-taniborbactam and FDC-xeruborbactam.Complimentary or enhancer effects of PBP2 inhibition with zidebactam or durlobactam also merit further investigation. 23inally, we propose that the role of microbiota-directed therapies like faecal microbiota transplantation could be explored for patients known to be colonized with extensively drug-resistant pathogens.

Table 1 .
Results of whole-genome sequencing and susceptibility testing of E. coli isolatesThe isolates exhibited 19 core SNP differences.Isolate-A was the baseline isolate causing bacteraemia, with no prior exposure to CZA-ATM or FDC.Isolate-B was collected from a surveillance rectal swab, following treatment with CZA-ATM.POD-30 (Supplementary Figures/Results).Interestingly, the abundance of E. coli during CZA-ATM therapy increased, which may be due to selection pressure to the commensal stool harbouring an E. coli population that had developed high-level resistance to ATM-AVI.Further studies are needed to longitudinally characterize the stool microbiota in CRE infection after transplantation and determine the variables associated with acquisition and resolution of CRE colonization.These studies are currently underway at our centre.
a New mutations that emerged following CZA-ATM therapy.Haidar et al.to